Project Summary Topoisomerase I (TOP1) is a FDA-approved therapeutic target for late-stage cancers with a recently expanded therapeutic potential. Canonical TOP1 poisons, such as topotecan, kill rapidly dividing cells by preventing the release of TOP1 from DNA during its topoisomerase reaction to relax supercoiled DNA, leading to DNA breaks. However, it was recently discovered that TOP1 poisons also alter gene expression linked to autism spectrum disorders (ASD) and acute inflammatory response, among others, via a mechanism that is independent of DNA damage. Indeed, in addition to relaxing supercoiled DNA, TOP1 also regulates mRNA splicing and gene expression through its interaction with RNA polymerase II (RNAPII) and splicing factors. Therefore, new drugs that modulate the transcriptional function of TOP1 without causing DNA damage could greatly increase the therapeutic scope of TOP1, and provide treatment options for difficult diseases. However, the mechanisms linking TOP1 to mRNA regulation are not well established. Our recent discovery that SUMOylation of the lysine (K) residues 391 and 436 of TOP1 regulates its transcriptional function provides a crucial insight that will accelerate mechanistic advances. Due to this new insight, we are well-positioned to uncover the detailed molecular mechanism linking TOP1 and mRNA regulation. This proposal will first test a novel hypothesis that TOP1 K391/K436 SUMOylation regulates RNAPII movement and the coupling of RNAPII and spliceosome to facilitate gene transcription (Aim 1). We will evaluate the transcriptional landscape and splicing patterns, including the presence of splice variants that are dependent on TOP1 K391/K436 SUMOylation. We will identify additional cellular processes that may be altered by the inhibition of the TOP1 transcriptional function to uncover its full potential for new disease treatments (Aim 2). We will also develop a novel molecular strategy to disrupt the effects of TOP1 on mRNA regulation. This novel strategy will facilitate drug development for a wide range of disorders, including inflammation-induced sepsis and ASD. Finally, in addition to facilitating transcription, K391/K436 SUMOylation also suppresses TOP1 topoisomerase activity. Because the sensitivity to TOP1 poisons positively correlates with the level of TOP1 topoisomerase activity in cells, we will test an exciting possibility that transiently blocking TOP1 SUMOylation by this SUMOylation inhibitor could hypersensitize cells to TOP1 poisons during cancer chemotherapy (Aim 3).